The mechanism of x-ray/alkylating agent induced leukemia is unknown. Chronic exposure of murine long-term bone marrow cultures or clonal Interleukin-3 (IL-3) dependent multipotential hematopoietic progenitor cell lines to one-ten-millionth to one one-hundred thousandth molar L-Phenylalanine Mustard (L-PAM) over 24 months in vitro induces variant cell lines with abnormalities including stable marker chromosomes, ten-thousandfold decreased IL-3 growth requirement, and altered differentiation capacity to erythroid/basophil/neutrophil lineages, but with no detectable in vivo leukemogenicity. In contrast, one-ten-millionth to one one-hundred thousandth L-PAM exposure or 50-500 Gy x-irradiation of purified marrow stromal cultures or a clonal stromal cell line induces colony formation by the same IL-3 dependent lines in agar overlay at -one-hundred thousand in the absence of detectable IL-3 in the system. Of 300 such individually removed and subcultured colonies, 25 were IL-3 independent and 3 of the 25 produced leukemia in vitro. Continuous exposure of overlaid hematopoietic target cells for 14-21 days was required to induce detectable transformation by this leukemogenic stromal factor (LSF). We now propose to elucidate the stromal cell phenotype(s) involved and the mechanism of humoral indirect transformation of hematopoietic stem cells by L-PAM treated or x-irradiated marrow stromal cells. Methods will include protein biochemistry; long-term bone marrow cultures; purified stromal and hematopoietic stem cell cultures from; control, previously irradiated or alkylating agent treated mice; assays for granulocyte-macrophage progenitor cells (GM-CFUc), pluripotential hematopoietic stem cells by reconstitution assay, spleen colony assay (CFUs), and assays for leukemogenecity of clonal cell lines in vivo. These studies should help elucidate the mechanism of irradiation and alkylating induced leukemia in patients surviving combination chemotherapy and irradiation therapy for malignancy.